Gene expression can be regulated both transcriptionally, at the DNA level, and post-transcriptionally, at the RNA level. The goal of this project is to understand how Drosophila RNA binding proteins act to regulate gene expression. We study a class of proteins that contain RNA recognition motif (RRM) domains, which have been found in many proteins involved in post-transcriptional regulation, and have been shown to mediate sequence-specific binding to RNA targets. We use a combination of genetic, biochemical and molecular approaches to analyze the functions of several Drosophila RRM proteins. Three of the proteins (HRB87F, HRB98DE and CAZ) are homologs of vertebrate nuclear pre-mRNA binding proteins, which associate with newly synthesized mRNA and affect its splicing and transport. Work over the past year has focussed on the identification and analysis of new mutations in each of these genes. One of the mutations in Hrb98DE is a premature stop codon that truncates the protein after the first RRM, and another is a small deletion that may remove much of the coding region for the N-terminal half of the protein. Both mutants are homozygous viable, indicating that HRB98DE is not essential. Similar results have been obtained for HRB87F. However, the double Hrb87F-Hrb98DE mutant is homozygous lethal. This synthetic lethality is seen with several combinations of Hrb mutants, and is specific for the Hrb genes, for mutations in other pre- mRNA binding proteins do not show this effect. We have recently identified mutations in the caz hnRNP gene, and in contrast to the Hrb mutants, these are lethal. Detailed analyses of their phenotypes are underway. In collaboration with the Mlodzik group at the EMBL, we are studying mutations in the Nts gene, which cause abnormal eye development, and sterility in both sexes. NTS contains one RRM and a region that resembles a zinc finger. Although Nts produces multiple transcripts, anti-NTS antibodies detect a single 75 kDa protein on Western blots. This protein is present in the cytoplasm, is detectable throughout development, and is particularly enriched in ovaries. Genetic interaction studies implicate NTS in signal transduction during eye development.